Detection of Listeria Species by Conventional Culture-Dependent and Alternative Rapid Detection Methods in Retail Ready-to-Eat Foods in Turkey

Foodborne pathogens, like Listeria monocytogenes, continue to inflict substantial financial losses on the food industry. Various methods for detecting Listeria in food have been developed and numerous studies have been conducted to compare the different methods. But, in recent years, new Listeria species have been identified, and currently the genus comprises 26 species. Therefore, it would be a more accurate approach to re-evaluate existing detection methods by considering new species. The present investigation involved the analysis of 42 ready-to-eat (RTE) foods, encompassing a variety of food categories, such as mezes, salads, dairy products, and meat products, with the aim of ascertaining the presence of Listeria. Among the traditional culture-dependent reference methods, the ISO 11290 method was preferred. The process of strain identification was conducted with the API Identification System. Furthermore, to ascertain the existence of L. monocytogenes and Listeria spp., the samples underwent additional analysis employing the VIDAS Immunoassay System, ELISA, and RT-PCR methodologies. Thus, four alternative approaches were employed in this study to compare not only the different methods used to determine Listeria while taking into account the newly identified Listeria species, but also to assess the compliance of retail RTE food items with microbiological criteria pertaining to the genus Listeria. Based on the conducted analyses, L. monocytogenes was conclusively determined to be present in one sample. The presence of Listeria spp. was detected in 30.9% of the samples, specifically in Turkish cig kofte, sliced salami, and salads.


Introduction
The genus Listeria is a highly significant group of microorganisms within the food industry.Currently, it comprises 26 validly published species, plus one species that doesn't yet have a valid status.Six species belonging to this genus have been known for a long time and include Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, Listeria welshimeri, and Listeria grayi.Since its initial discovery in 1926, the genus consisted of only eight species till the year 2010 [1,2].Since this date, a multitude of novel species have been discovered, leading to a rapid expansion of the genus.The traditional properties of Listeria, such as its ability to survive at 4°C, have lost their universality due to the discovery of new species.With all of this new information about Listeria, the genus has been subdivided into two clades: sensu stricto (L.monocytogenes and similar species to L. monocytogenes in terms of characteristics) and sensu lato (species more distantly related to L. monocytogenes).On the other hand, despite the discovery of new Listeria species, whether from the sensu stricto or sensu lato clade, within the genus, type of food, it is crucial to prevent contamination throughout the entire process, from production to consumption.Furthermore, RTE foods with a neutral or moderately low pH and relatively high a w values are considered to support the growth of L. monocytogenes.Many countries like the US, New Zealand, Australia, and Turkey currently have a "zero tolerance" approach for all RTE food.However, despite all the precautions and legal regulations, listeriosis outbreaks due to consumption of RTE foods are still observed all over the world [4,7,[10][11][12].
Contemporarily, various methods for detecting Listeria spp. or L. monocytogenes in food have been developed.Culture-dependent traditional methods, recommended by entities like the U.S. Food and Drug Administration (FDA), the International Organization for Standardization (ISO), and the Association of Official Analytical Chemists (AOAC), are still considered the gold standard.The FDA's Bacteriological Analytical Manual (BAM), ISO 11290, and the AOAC's official method are routinely used to isolate and identify Listeria species.However, since culture-dependent methods are time-consuming, alternative and rapid detection methods based on different approaches have also been developed over time.These methods can be categorized into immunological, spectroscopic, and molecular methods, as well as microfluidic system-, biosensor-and phage-based methods [13][14][15].Immunological methods are based on specific antigen-antibody binding.Enzyme-linked immunosorbent assay (ELISA), enzyme-linked fluorescent assay (ELFA), thermal flow immunoassay and immunomagnetic separation are among the most used immunological methods [13].Simple polymerase chain reaction (PCR), multiplex PCR, real-time PCR (RT-PCR), real-time nucleic acid sequence-based amplification, oligonucleotidebased microarray, and loop-mediated isothermal amplification are among the most used molecular detection methods.Near infrared spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and raman spectroscopy are among the most used spectroscopic methods.In recent times, in addition to these methods, sensor systems have also been developed to detect Listeria, and these include optical, electrochemical, piezolectic, and cell-based sensors [14][15][16].
Numerous studies have been conducted to compare existing systems with one another.Various methodologies used to identify Listeria species exhibit distinct merits and drawbacks relative to one another.However, it is worth noting that a majority of these investigations were conducted prior to the identification of novel Listeria species.Consequently, the conducted analyses did not account for newly discovered species.
In Turkey, foods called 'meze' , which are in the RTE food category and consumed with alcoholic beverages, such as raki, or eaten at the beginning of the meal, are very popular.Foods in this category are sold in supermarkets and public markets.In the current study, 42 RTE foods (mostly meze and salads) were analyzed to determine the presence of Listeria spp.and L. monocytogenes by using four different methods, including classical method, ELISA, ELFA, and RT-PCR.The aim herein was to evaluate whether retail RTE food products in supermarkets and public markets comply with microbiological criteria in terms of L. monocytogenes, and also to compare different methods used to determine Listeria, considering both sensu stricto or sensu lato clades.

Pilot Study
In the first stage of the study, commercially sold pasteurized milk, which is known not to contain Listeria species, was contaminated in a controlled manner, and all analysis methods used in the study were tested.Purchased milk was divided into 3 parts under sterile conditions and Listeria monocytogenes 4b ATCC 19115 was added at concentrations of 1 CFU/ml, 10 CFU/ml, and 100 CFU/ml, respectively.Then, all analyses described in detail below were carried out simultaneously with these controlled contaminated samples.

Sample Collection
A total of 42 RTE foods including various mezes, salads, meat, and dairy products were randomly purchased from supermarkets and public markets in Eskişehir, Turkey, over a three-month period.As soon as they were purchased, samples were aseptically transported to the laboratory in sterile bags under refrigerated conditions, and then immediately transferred to the pre-enrichment medium.Information obtained from the sellers about the RTE foods used in the study is given in Table 1.

Isolation and Identification of Listeria with Conventional Method
In the present study, the EN ISO 11290-1 [17] guidelines were followed for the isolation of Listeria strains by culture-dependent methods.Then, selected colonies were identified by using the API Listeria Identification System.Half-Fraser broth (225 ml medium in sterile stomacher bags), Fraser broth (10 ml medium in sterile glass test tubes), API Listeria Tests (10 300) were purchasedfrom BioMeriux, Turkey.Agar Listeria according to Ottaviani and Agosti (ALOA), Palcam Agar, and TSA-YE agar were purchased from Merck, Turkey.The details of the process steps are as follows: For the pre-enrichment step, a 25 g sample was added to 225 ml of Half-Fraser broth medium and after homogenization in a stomacher (Smasher BioMerieux, France) for 1 min, the mixture was incubated for 25 ± 1 h at 30°C.Then, for the enrichment step, a 0.1 ml sample was collected and transferred into 10 ml Fraser broth medium and incubated for 24 ± 2 h at 37°C.At the end of the incubation period, samples were inoculated onto ALOA and Palcam Agar plates and incubated for 48-72 h at 37°C.Following that, typical Listeria colonies were selected, examined by gram-staining, and grown on TSA-YE agar for the API Listeria test.After 24-48 h incubation on TSA-YE agar at 37°C, colonies harvested with a sterile loop were transferred to the API suspension medium.The density of suspension medium was adjusted to equal BioMerieux McFarland 1.Then, after distributing approximately 3 ml of distilled water to the bottom of an incubation box and placing the strip inside it, the prepared suspension medium was distributed to each tube.The lid of the incubation box was closed, and the February 2024  Vol.34  No. 2 test kit was incubated for 18-24 h at 37°C.At the end of the incubation period, a drop of ZYM B reagent was added to the DIM test and after 3 min, all reactions were read in accordance with the reading chart.For the evaluation, a 4-digit numerical profile was obtained by using the result sheet according to the administrator's instructions.Species determinations were made by entering the obtained numerical profile into the apiweb database (V2.0) optimized by the company.

Listeria spp. Detection with ELISA
To determine Listeria spp. in food samples by the ELISA method, a Tecra Listeria Visual Immunoassay (Tecra Diagnostic) test kit was used.Since this commercially available kit requires pre-enrichment and enrichment steps prior to analysis, the samples prepared for determination by classical method as described before were also used in this study.At the 24th h of incubation in the enrichment step in Fraser broth medium, 1 ml samples were taken and used for ELISA analysis.Analysis was performed using the solutions provided with the kit, according to the administrator's instructions.Briefly, a 50 μl sample additive and a 1 ml sample were added to a test tube and kept in a water bath for 15 min at 100°C.Meanwhile, for each sample, 1 well was placed in the holder.Also 2 wells were added in the holder for positive and negative controls.For each sample, 200 μl of heat-treated sample was transferred into individual wells.Then, 200 μl of positive control solution and 200 μl of negative control solution were added to positive and negative control wells, respectively.All wells were covered with parafilm and incubated for 30 min at 37°C.After that, the wells were inverted to discard the content and then washed 3 times with intensive washing solution.Then, 200 μl of conjugate was added to the cells.All wells were covered with parafilm, and after incubation for 30 min, the content was discarded again and washed 4 times.In the next step, 200 μl of substrate was added to the cells and kept for 15 min at room temperature.At the end of the incubation period, both the color Table 1.Types of food included in the study and some information about them.

Sold in portions according to customer demand and unpackaged
Turkish cig kofte Turkish cig kofte (raw meatball) is prepared by kneading ground meat and bulgur with tomato paste, red pepper paste, pomegranate syrup and various spices for about an hour with the addition of hot water.
Authentic Turkish Ezme Authentic Turkish Ezme is prepared by mixing the peeled and very finely chopped tomatoes (red and sun ripened), onions, green pepper and red pepper with paprika, sumac, salt, chopped parsley and mint.

Tarator
Tarator is a kind of mezze made by mixing bread crumbs, walnuts olive oil and lemon juice, olive oil, garlic and vinegar.

Humus
Hummus is prepared by mixing tahini, chickpeas, garlic, salt, cumin, red pepper, lemon juice and olive oil, until a thick-smooth paste.

Haydari
Haydari is one of the most popular mezze in Turkey prepared by well mixing yoghurt, fullfat feta cheese (mashed), dill (finely chopped) garlic (grated), mint and salt.

Lentil meatballs
Lentil meatballs is prepared by kneading boiled red lentils and fine bulgur with tomato paste, finely chopped parsley, finely chopped green onion, various spices, salt and onions sautéed in olive oil.

Sold in portions according to customer demand and unpackaged
Russian Salad Italian Salad American Salad In Turkey, these salads are basically prepared by adding mayonnaise and yogurt to a mixture of potatoes (boiled and chopped), carrots (boiled and chopped), peas (boiled), canned corn and thinly sliced pickles.The difference between 3 salads in the food sector in Turkey is generally as follows.American salad is made with vegetables only.Russian salad includes cold cuts of meat in addition to vegetables.Italian salad, on the other hand, includes julienne-cut salami in addition to vegetables.

Mushroom Salad
In Turkey this salad is mostly prepared by mixing boiled mushrooms, roasted red pepper (chopped) pickled gherkins (chopped), dill, canned corn, lemon juice, pomegranate syrup salt and olive oil.

Roasted Eggplant Salad
In Turkey this salad is prepared by mixing the roasted and peeled eggplant (cut cubes), roasted and peeled bell pepper (finely sliced) tomato (finely chopped), onion (finely chopped), parsley (finely chopped), lemon juice, salt, black pepper, paprika and olive oil.

Ham Chicken doner Sliced Sausage
These products, which can be produced by different brands, are sold packaged and sliced.
Dairy products Yoghurt Cream Curd cheese These products, which can be produced by different manufacturers, are sold in portions/ grams according to customer demand and unpackaged change in the wells was evaluated visually using the color chart (1 and 2 negative, 3, 4 and 5 positive) and the results were recorded and the absorbance at 414 nm was recorded with an ELISA reader.

L. monocytogenes Detection with ELFA
To determine L. monocytogenes in food samples by the ELFA method, a VIDAS Immunoassay System (BioMerieux) was used.This system is similar to the determination of Listeria spp. with the ELISA method, since it also requires pre-enrichment and enrichment steps prior to analysis and the samples in the enrichment step, prepared for determination by the classical method, were used.At the 24th h of incubation in the enrichment step in Fraser broth medium, 500 μl samples were taken and transferred to a Listeria monocytogenes II (LMO2, BioMerieux) test kit.In addition to the samples, standard samples called S1, a positive control called C1, and a negative control called C2 were added to the wells of the strip in the order of identification on the VIDAS device.Then, the strips were inserted into the device and the test was conducted according to the administrator's instructions.

Listeria spp. Detection with RT-PCR
For determination of Listeria spp. in food samples by RT-PCR, in the first step DNA extraction was performed using a Food DNA Extraction Kit (Genoks GFJ-DNA 022, Turkey) and used as a template for RT-PCR analysis.For the extraction, as in the other determination test, the samples in the enrichment step prepared for determination by the classical method were used.At the 24 th h of incubation in the enrichment step in Fraser broth medium, a 2 ml sample was taken and centrifuged at 12 rpm for 2 min.The obtained pellet was used for DNA isolation.All process steps were carried out with the ready-made solutions included in the kit according to the instructions of the administrator.
For RT-PCR, a Listeria spp.RT-PCR kit (Genoks GFJ-420) was used.Texas Red dye was used for scanning.Analysis was carried out, as recommended by the manufacturer, in 50 μl reaction volume containing 35 μl of RT-PCR master mix, 5 μl of Listeria spp.-specific probe, 5 μl template DNA, and 5 μl dH 2 O. Analysis conditions consisted of initial denaturation for 2 min at 94°C, followed by 40 cycles of denaturation for 45 s at 94°C, annealing for 45 s at 60°C, and extension for 45 s at 72°C.

Statistical Analysis
The data obtained from the study were analyzed by using the SPSS 23.0 program.Man-Whitney test, one of the non-parametric tests, was used to compare the Listeria detection methods, including conventional culturedependent methods, ELISA, and RT-PCR.

Results and Discussion
To determine the presence of pathogenic microorganisms in food, numerous alternative detection methods have been developed over time.Among these methods, some immunological techniques such as ELISA and ELFA, and some molecular methods like RT-PCR, are considered widely accepted due to their ease of application, relative cost-effectiveness, and long-standing use.As a result, these techniques have been accepted as the most commonly preferred methods along with the traditional culture-dependent methods [14,15].Therefore, in the current study, in addition to the traditional culture-dependent reference method, EN ISO 11290-1, these three methods were chosen based on their widespread acceptance and practicality.
In the first stage of the present study, pasteurized milk sample was contaminated in a controlled manner and preliminary trials were carried out for the four different analysis methods, which included the classical method, ELISA, ELFA, and RT-PCR.As a result of the preliminary trials, it was observed that correct results were obtained with all methods used, even when the pasteurized milk sample was contaminated with L. monocytogenes at a concentration of 1 CFU/ml.Thus, the methods were confirmed to have been applied correctly.The study design and results of the preliminary trials were presented in Fig. 1.After the preliminary studies, 42 RTE foods sold in supermarkets and public markets in Eskişehir, Turkey was analyzed for the presence of Listeria species using more than one method and the results were presented in Table 2.
Today, a wide variety of sensitive, reliable, and validated methods are available for the determination of Listeria in foods.Of these methods, FDA BAM, ISO 11290, and the AOAC's official methods are currently accepted as reference methods worldwide, and all other methods are validated through comparison with these methods [15,18].Even so, the reference methods mentioned also have some limitations.Besides being time-consuming and laborious, one of the main problems with these methods are the possibility of false-negative results.It is known that various sensu stricto species, such as L. seeligeri and L. ivanovii, cannot be detected from time to time if the samples contain low levels and/or damaged cells [3,14].Moreover, several recent studies have shown that some newly identified Listeria sensu lato species may not be determined by the ISO method [19].In the present study, the presence of Listeria was detected in 13 food samples by the ISO 11290 method.On the other hand, ELISA and RT-PCR analyses showed the presence of Listeria in 18 food samples, and the samples containing Listeria spp.showed identical results with the two methods.As a result of the statistical analysis, no significant difference was found between the conventional culture-dependent method, ELISA, and RT-PCR (p > 0.05).In the analysis performed by the conventional method, the presence of Listeria could not be detected in 5 of these 18 samples mentioned.The other 13 samples agreed with the samples in the ELISA and RT-PCR analyses.As described above, a false-negative result may have been seen for the 5 samples by the ISO 11290 method, due to either insufficient cell presence, cell damage, or the presence of Listeria sensu lato species.Furthermore, similar results can be obtained in the case of contamination with viable but non-culturable (VBNC) bacteria.On the other hand, the difference in the results obtained from the conventional culture-dependent and alternative methods in the present study may be due to other reasons.Since Listeria detection by alternative and rapid detection methods like ELISA and RT-PCR is independent of strain viability, dead cells can also be detected with these systems [13,20].Similarly, in the present study, it was possible to find dead cells and get false-positive results in 5 samples by ELISA or RT-PCR.
The Tecra Listeria VIA test kit used in this study is a sandwich configuration-based ELISA system and is suitable for detecting Listeria spp. in foods.This kit has a very high accuracy rate and has been approved by the AOAC as a colorimetric polyclonal immunoassay screening method.However, various studies have shown that the system can give false-positive and false-negative results, albeit rarely.Noah et al. [21], to evaluate commercial ELISA kits, analyzed 178 samples using both commercial Tecra Listeria VIA kits and BAM reference methods.At the end of the study, the researchers determined the presence of Listeria in 38 and 40 samples, by the BAM and Tecra methods respectively, and although they gave more false-positive results, the researchers recommended the Tecra Listeria VIA kit as an alternative to the BAM method as rapid screening procedures.Vanderlinde and Grau [22] investigated the presence of Listeria spp. in 170 samples and reported 2 false-positive reactions in their analysis by Tecra Listeria VIA test.
The possibility of false-positive and false-negative results is not seen only in immunological methods such as ELISA.Similar situations are also observed in nucleic acid-based methods, such as RT-PCR, whose validity and reliability have been proven.Aznar and Solis [18] screened the presence of L. monocytogenes in 225 food samples by three methods and reported 22, 23, and 60 positive samples by ISO 11290-1, VIDAS LMO, and PCR methods, respectively.Netschajew et al. [23] evaluated RT-PCR and classical culturing methods for detecting L. monocytogenes in 50 vacuum-packed meat products and reported positive results in only a few samples by conventional methods; however, they detected the presence of L. monocytogenes in 32 samples by RT-PCR.Barbau-Piednoir et al. [24] reported that they developed a SYBR Green qPCR system that works with 98% accuracy for the detection of Listeria spp. in foods.
In the reference methods, identification of presumptive Listeria colonies is performed by morphological, biochemical, and physiological tests.In these types of analysis, after the isolation to shorten the total analysis time, various commercial rapid identification tests like the API Listeria identification system are often preferred [13,14,18].Similarly, in the present study, after purification of Listeria strains from ALOA or Palcam agar medium by the conventional method, the identification of strains was performed with the API Listeria test.As a result, 6 strains were identified as L. innocua, 2 were L. grayi, 1 was L. seeligeri, 1 was L. ivanovii, and 1 strain was L. monocytogenes.The strains found in samples 2 and 13 could not be identified.Although the API Listeria kit is a highly accurate method, in the test, the results are evaluated according to the color change in the first stage, based on qualitative observation.This situation increases the margin of error during the evaluation of the results and may lead to misidentification or failure to identify strains [25].Carlin et al [2] reported that Listeria API analysis misidentified L. swaminathanii as L. monocytogenes.Aznar and Solis [18] isolated possible Listeria strains from 225 food samples with the ISO-11290 method, and identified the strains with the API Lis system.After confirming the identity of the strains with specific PCR, the researchers reported 5 false-positive and 7 false-negative identification results with API.
Since L. monocytogenes is the only species in the genus Listeria to be considered a human pathogen, most of the developed methods are for the direct detection of L. monocytogenes in foods.VIDAS Immunoassay system is  suitable for determining both Listeria spp.and L. monocytogenes through different kits developed [26].VIDAS LMO2 kit has been developed for the direct detection of L. monocytogenes and validated by ANFOR certification [27].Although this system is now considered an effective alternative to traditional methods [28,29], the accuracy of the system is recognized as not always 100% and it is recommended that positive results be confirmed with conventional methods [26,30].In a study that analyzed 295 foods, 11 false-positive results were obtained in the assay of L. monocytogenes with VIDAS LMO2 kits and researchers reported that the specificity of VIDAS LMO2 was 0.96 and the sensitivity 0.73 [30].A similar result was also observed in the present study.One strain isolated from one sample (sample no.28) was identified as L. monocytogenes by the API Lis kit.However, analysis with VIDAS Immunoassay system showed the presence of L. monocytogenes in two samples (samples no. 15 and 28).
Colonies in sample no.15, whose presence of L. monocytogenes was determined by VIDAS, were identified as L. innocua by API test.For this study, with the available data, it is not possible to say which test results are correct.False-positive results may be obtained with VIDAS analysis, and species can be misidentified by API test.Moreover, there is a third possibility; selective media preferred for analysis and Listeria species present in a sample can affect the detection sensitivity.It has been reported in the literature that selective agents such as acriflavine added to Fraser broth may have harmful effects such as stress or cell damage on L. monocytogenes.In the preenrichment step of the ISO 11290 method, an attempt to solve this problem was made by adding a semiconcentration selective agent into Half-Fraser broth medium.Besides, in Fraser broth medium, Listeria innocua has been shown to inhibit the development of L. monocytogenes 1/2a [14,20].Similarly, in this study, the presence of L. innocua in sample no. 15 may have suppressed the isolation of L. monocytogenes by culture-dependent methods, but positive results may have been obtained with this system since the VIDAS system detects antigens rather than living cells.All the results obtained from the four different methods were evaluated together, and in the present study, 30.9% of analyzed RTE foods with certainty contained Listeria species, especially L. innocua.Additionally, it is noteworthy that Listeria species are found especially in Turkish cig kofte from mezes, sliced salami from meat products and generally in salads.The presence of L. monocytogenes is certain in only one of the 42 RTE foods.Compared with existing literature data, in the present study, the rate of L. monocytogenes is lower.Bilgin et al. [31] analyzed 100 samples of salad and appetizers from retailers in Istanbul and reported that they found L. monocytogenes in 9% of the samples.Bustamante et al. [32] analyzed 400 RTE artisanal food samples requiring minimal or moderate process and reported L. monocytogenes in 7.5% of samples.Łepecka et al. [12] analyzed 30 different RTE salads sold in Polish markets and reported L. monocytogenes in 33.3% of the salads.From the perspective of food hygiene, it should be noted that the presence of Listeria species may indicate potential risk of contamination with L. monocytogenes [13,20].Therefore, even if the L. monocytogenes rate was found to be low, it is clear that these products are risky for Listeria.

Conclusion
The conventional culture-dependent techniques employed for the identification of Listeria species continue to be regarded as the benchmark method.Nonetheless, the limitations of culture-dependent methods, particularly in detecting sensu lato Listeria species, and the incapability to detect non-viable/damaged cells, may lead to differences in the results obtained from culture-dependent and other frequently preferred methods.As in this study, the available data obtained from one culture-dependent method and one alternative method like ELISA can be insufficient, and further analyses may be needed to determine which of the different methods used gives the correct result.Due to the risk of misleading results, within species-based identification, it is crucial to use molecular-based techniques to validate the identification of strains following isolation.This type of approach can ensure a definitive and unequivocal confirmation, eliminating any uncertainties.In this context, there is still a need to develop fast and reliable methods for detecting all Listeria species and differentiating them at the species level.

Fig. 2 .
Fig. 2. Results of preliminary trials.(A) The 4-digit numerical profile and the identified species resulting from the identification of colonies with the API test kit, after isolation with conventional method according to EN ISO 11290-1 standards from a pasteurized milk sample to which Listeria monocytogenes 4b 19115 was added at a concentration of 1 CFU/ml.(B) ELIZA preliminary study results.Wells from left to right;1: positive control, 2: negative control, 3: pasteurized milk contaminated L. monocytogenes at a concentration of 1 CFU/ml, 4: pasteurized milk contaminated L. monocytogenes at a concentration of 10 CFU/ml, 5: pasteurized milk contaminated L. monocytogenes at a concentration of 100 CFU/ml.(C) VIDAS preliminary test results.Component A contains standards, positive control and negative control, while component B contains samples to be tested.In component B; B1 pasteurized milk contaminated L. monocytogenes at a concentration of 1 CFU/ml, B2: pasteurized milk contaminated L. monocytogenes at a concentration of 10 CFU/ml, B3: pasteurized milk contaminated L. monocytogenes at a concentration of 100 CFU/ml.(D) RT-PCR preliminary test results; The graph showing a logarithmic increase from the 5th cycle is the positive control.The graph showing a logarithmic increase from the 20th cycle is the reaction established with gDNA isolated from contaminated milk, and the graph showing no increase is the reaction established with E. coli gDNA.